In vivo control of microorganisms
Antiviral agents
Virology; the study of viruses
(or, lifestyles of the small and nasty)
Viruses have one major characteristic in common: they are obligate intracellular
parasites.
Viruses are UNABLE to grow and reproduce outside of a living cell. No virus
is able to produce its own energy (ATP) to drive macromolecular synthesis.
However, in many other respects, they are a highly diverse group.
The size of viruses
Strategies for virus survival
•
Finding and getting into a host cell. As viruses are obligate parasites they must find the
right type of cell for their replication, they must invade that cell and get their genome to the
site of replication.
•
Making virus protein. All viruses are parasites of translation. The virus must make mRNA
(unless it has a + sense RNA genome already). Strategies must exist to synthesize mRNA.
•
Making viral genomes. Many viral genomes are copied by the cell’s synthetic machinery in
cooperation with viral proteins.
•
Forming progeny virions. The virus genome, capsid (and envelope) proteins must be
transported through the cell to the assembly site, and the correct information for assembly
must be pre-programmed.
•
Spread within and between hosts. To ensure survival the virus must propagate itself in
new cells.
•
Overcoming host defences.The host defends itself against “nonself”. Viruses have
evolved ways to fight back.
Three problems every virus must solve
• 1
• 2
• 3
How to reproduce during its “visit”
inside the cell. How to a) copy its genetic
information and b) produce mRNA for protein
production
How to spread from one individual to
another
How to evade the host defenses. This
need not be complete.
• Viral diseases are the (usually unintended)
consequences of the way each virus has chosen
to solve these three problems.
How are viruses named?
• Based on:
- the disease they cause
poliovirus, rabies virus
- the type of disease
murine leukemia virus
- geographic locations
Sendai virus, Coxsackie virus
- their discovers
Epstein-Barr virus
- how they were originally thought to be contracted
dengue virus (“evil spirit”), influenza virus (the “influence” of bad air)
- combinations of the above
Rous Sarcoma virus
Virus Classification
Taxonomy from Order downward (three orders now recognized)
•Family often the highest classification. Ends in -viridae.
•Many families have subfamilies. Ends in -virinae.
•Bacterial viruses referred to as bacteriophage or phage (with a few exceptions).
Examples
family Myoviridae
genus T4-like phages
type species Enterobacteria phage T4
family Herpesviridae, subfamily Betaherpesvirinae
genus Muromegalovirus
type species Murine herpesvirus 1
The Baltimore classification system
Based on genetic contents and replication strategies of viruses. According to the
Baltimore classification, viruses are divided into the following seven classes:
1. dsDNA viruses
2. ssDNA viruses
3. dsRNA viruses
4. (+) sense ssRNA viruses (codes
protein)
5. (-) sense ssRNA viruses
6. RNA reverse transcribing viruses
7. DNA reverse transcribing viruses
directly for
where "ds" represents "double strand" and "ss" denotes
"single strand".
RNA viruses
From Principles of Virology Flint et al ASM Press
DNA viruses
From Principles of
Virology Flint et al
ASM Press
Structural Classes
•Icosahedral symmetry
•Helical symmetry
•Non enveloped (“naked”)
•Enveloped
Icosahedral capsids
a) Crystallographic structure of a
simple icosahedral virus.
b) The axes of symmetry
Enveloped helical virus
Enveloped icosahedral virus
Typical infectious cycle
1.
Attachment
2.
Penetration
3.
Uncoating
4.
Transcription and/or translation
5.
Replication
6.
Assembly
7.
Release
Virus recognition, attachment, and entry
•Specific viral receptor
•Co-receptor
•Receptor-mediated endocytosis
•Fusion of the viral membrane at the cell surface
RECEPTOR VIRUS
ICAM-1
polio
CD4
HIV
acetylcholine
rabies
EGF
vaccinia
CR2/CD21
EpsteinBarr
herpes
HVEM
Sialic acid
Influenza,
reo, corona
Receptor-mediated endocytosis of poliovirus
The two basic modes of entry of an
enveloped animal virus
Drugs that Prevent the Virus from Entering or
Leaving the Host Cells
• Amantadine – interferes with replication of influenza A by
inhibiting the transmembrane M2 protein that is essential for
uncoating the virus.
- Has a narrow spectrum; so, flu vaccine is usually preferable.
• Zanamivir – inhibits both influenza A and B neuraminadase.
Decr duration of symptoms if given within 48 hr of the onset
of symptoms. Prophylactic in healthy adults.
• Immunoglobulins – Human Ig contains specific Abs against
superficial Ags of viruses  can interfere with their entry into
host cells. Protection against hepA, measles, and rubellla
(German measles).
Drugs that Inhibit Nucleic Acid Synthesis
Nucleoside and Nucleotide Analogs
• Acyclovir- used to treat genital herpes
• Cidofovir- used for treatment of
cytomegaloviral infections of the eye
• Lamivudine- used to treat Hepatitis B
Antiviral Drugs
Nucleoside and Nucleotide Analogs
Figure 20.16a
Acyclovir
• HSV and VZV contain a thymidine kinase (TK) that 
acyclovir to a monophosphate phosphorylated by
host cell enzymes to acycloguanosine triphosphate,
which inhibits viral DNA pol and viral DNA synthesis.
• Selectively toxic (TK of uninfected host cells activates
only a little of the drug).
• Viral enzymes have a much higher affinity than the
host enzymes for the drug.
• Effective against HSV, but does not eradicate them.
• Need high doses to treat shingles.
Ganciclovir
• Quite toxic (neutropenia) –so, given only for
severe CMV infections in immunosuppressed
patients.
• CMV is resistant to acyclovir because it does
not code for TK.
Other enzyme inhibitors
• Zanamivir (Relenza) and Oseltamivir
phosphate (Tamiflu)- inhibitors of the enzyme
neuominidase
– Used to treat influenza
• Indinavir- protease inhibitors. Inhibit the
synthesis of essential viral proteins (e.g., RT)
by viral-specific proteases.
Interferons
• Cells infected by a virus often produce
interferon, which inhibits further spread of the
infection
• Alpha-interferon - drug for treatment of viral
hepatitis infections
Antiviral Drugs
Enzyme Inhibitors
• Protease inhibitors
– Indinavir
• HIV
• Inhibit attachment
– Zanamivir
• Influenza
• Inhibit uncoating
– Amantadine
• Influenza
• Interferons prevent spread of viruses to new
cells
• Viral hepatitis
Treatment of Herpesviruses
Varicella-zoster,
Cytomegalavirus,
Herpes simplex
Anti-metabolites
•
“False” DNA building blocks or nucleosides. A nucleoside
consists of a nucleobase and the sugar deoxyribose.
• In antimetabolites, one of the components is defective. In the
body, the abnormal nucleosides undergo bioactivation by
attachment of three phosphate residues
• Acyclovir has both specificity of the highest degree and
optimal tolerability, because it undergoes bioactivation only in
infected cells, where it preferentially inhibits viral DNA
synthesis.
Acyclovir
•
A virally coded thymidine kinase (specific to H.simplex and
varicella-zoster virus) performs the initial phosphorylation
step; the remaining two phosphate residues are attached by
cellular kinases.
•
Acyclovir triphosphate inhibits viral DNA polymerase
resulting in chain termination.
It is 30-fold more potent against the virus enzyme than
the host enzyme.
Acyclovir is active against herpes simplex and varicellarzoster virus.
It is rapidly broken down in cells, is orally active and is
relatively non-toxic systemically.
Acyclovir
Acyclovir is used to treat:
•
Herpes simplex infections (genital herpes, and herpes
encephalitis).
•
Chickenpox in immuno-compromised patients.
•
Prophylactically in patients
treated
with
immunosuppressant drugs or radiotherapy who are in
danger of infection by reactivation of latent virus.
•
Prophylactically in patients with frequent recurrences
of genital herpes.
Vidarabine
• Inhibits virally induced DNA polymerase more strongly than it
does the endogenous enzyme.
• Vidarabine is a chain terminator and is active against herpes
simplex, varicella zoster, and vaccinia are especially
sensitive.
• Its use is now limited to topical treatment of severe herpes
simplex infection. Before the introduction of the better
tolerated acyclovir, vidarabine played a major part in the
treatment of herpes simplex encephalitis.
• Its clinically used in treatment of immunocompromised
patients with herpetic and vaccinia keratitis and in
keratoconjunctivitis.
Treatment of respiratory virus infection
Influenza A & B
Respiratory suncytial virus (RSV)
Attachment Inhibitors
•
The primary antiviral mechanism of Amantadine and
Rimantadine is to block the viral membrane matrix
protein, which function as an ion channel that is required
for the fusion of the viral membrane with the cell
membrane.
•
Their clinical use is limited to Influenza A infection.
•
They are very effective in preventing infection if the
treatment is begun at the time of-or prior to- exposure to
the virus.
Attachment Inhibitors
•
Side effects of Amantadine are mainly associated with the
CNS, such as ataxia and dizziness.
•
While Rimantadine produce little CNS effect because it
does not penetrate the blood brain barrier.
•
Both should be used with caution in pregnant and nursing
women.
Neuroaminidase inhibitors
Oseltamivir and Zanamavir
Mechanism of action
• Viral neuraminidase catalyzes cleavage of terminal sialic
acid residues attached to glycoproteins and glycolipids, a
process necessary for release of virus from host cell surfaces.
•Neuraminidase inhibitors thus prevent release of virions
from infected cell
40
Neuroaminidase inhibitors
• Administration of neuraminidase inhibitors is a treatment
that limits the severity and spread of viral infections.
• Neuraminidase inhibitors are useful for combating influenza
infection:
zanamivir, administered by inhalation;
oseltamivir, administered orally.
• Toxicities
– Exacerbation of reactive airway disease by zanamavir
– Nausea and vomiting for oseltamivir
Antiretroviral agents
Antiretrovirals
• Currently implies a drug used to treat HIV
• Tenofovir- nucleotide reverse transcriptase inhibitor
• Zidovudine- nucleoside analog – inhibits RT of HIV and is only
used orally for AIDS.
- Activated by triple phosphorylation and then binds RT
(with100X affinity than for cellular DNA pols).
- Incorporated into the DNA chain, but lacks a 3’OH; so
another nucleoside cannot form a 3’-5’-phosphodiester bond
 DNA chain elongation is terminated.
-Severe adverse effects: anemia, neutropenia, myalgia,
nausea, and headaches.
• Stavudine, didanosine, zalcitabine – among other NRTIs.
• Nevirapine, efavirenz – Non nucleoside RTIs - denature RT.
Life Cycle of
HIV
HIV gp41-mediated fusion and enfuvirtide (T-20) action – Prohibits HIV entry
HIV Life Cycle
Step 1: Fusion
HIV
Step 3:
Integration
reverse
transcriptase
Step 5: Packaging
and Budding
Step 2: Transcription
Step 4: Cleavage
Azidothymidine (Zidovudin(AZT))
• It is a potent antagonist of reverse transcriptase, It is a
chain terminator.
• Cellular enzyme phosphorylate AZT to the triphosphate form
which inhibits RT and causes chain termination
• It is widely use in the treatment of AIDS (The only clinical
use).
• AZT is toxic to bone marrow, for example, it cause severe
anaemia and leukopenia In patient receiving high dose.
Headache is also common
Didanosine (Dideoxyinosine)
• Didanosine act as chain terminators and inhibitors of
reverse transcriptase because they lack a hydroxyl group.
• is phosphorylated to the
dideoxyadenosine triphosphate
active
metabolite
of
• It is used in the treatment of AIDS (second drug approved
to treat HIV-1 infection).
• They are given orally,
• and their main toxicities are pancreatitis, peripheral
neuropathy, GI disturbance, bone marrow depression.
Abacavir
• Abacavir is a guanosine analog (Figure 49–2) that is well absorbed
following oral administration (83%) and is unaffected by food. The serum
half-life is 1.5 hours. The drug undergoes hepatic glucuronidation and
carboxylation. Cerebrospinal fluid levels are approximately one third those
of plasma.
• Abacavir is often co-administered with lamivudine, and a once-daily, fixeddose combination formulation is available. Abacavir is also available in a
fixed-dose combination with lamivudine and zidovudine.
• High-level resistance to abacavir appears to require at least two or three
concomitant mutations and thus tends to develop slowly.
• Hypersensitivity reactions, occasionally fatal, have been reported in up to
8% of patients receiving abacavir and may be more severe in association
with once-daily dosing.
• All NRTIs may be associated with mitochondrial toxicity,
probably owing to inhibition of mitochondrial DNA
polymerase gamma. Less commonly, lactic acidosis with
hepatic steatosis may occur, which can be fatal. NRTI
treatment should be suspended in the setting of rapidly rising
aminotransferase levels, progressive hepatomegaly, or
metabolic acidosis of unknown cause. The thymidine analogs
zidovudine and stavudine may be particularly associated with
dyslipidemia and insulin resistance. Also, some evidence
suggests an increased risk of myocardial infarction in patients
receiving abacavir or didanosine; this bears further
investigation.
Non-nucleoside Non-competitive
RT inhibitors
(1) bind to viral RT, inducing conformational changes that result in
enzyme inhibition
(2) Combination therapy with AZT (resistant mutants rapidly
emerge, little use in monotherapy)
(3) Resistance mutations will be at different sites
Generic Name
Nevirapine
Trade Name
Viramune
Delavirdine
Rescriptor
Usual Dose
200 mg QD x14
days, then
200 mg BID
400 mg TID
Efavirenz
SustivaTM
600 mg QD
51
Non-nucleoside Non-competitive
RT inhibitors
Nevirapine Approved for AIDS patients, Good blocker of mother
to child transmission (perinatal - breast feeding)
• Single dose at delivery reduced HIV transmission by 50%
• Single dose to baby by 72 hours
NNRTI’s: Adverse Effects
RASH!!
CNS effects (e.g. sedation, insomnia, vivid dreams, dizziness,
confusion, feeling of “disengagement”)
52
Rash
Rash, usually a maculopapular eruption that spares the palms
and soles, occurs in up to 20% of patients, usually in the first
4–6 weeks of therapy.
Although typically mild and self-limited, rash is dose-limiting in
about 7% of patients. Women appear to have an increased
incidence of rash.
When initiating therapy, gradual dose escalation over 14 days
is recommended to decrease the incidence of rash.
Protease Inhibitors
•
HIV Protease Inhibitors; have significantly alter the course
of the HIV disease.
•
All are reversible inhibitors of HIV Protease-the viral
enzyme responsible for cleavage of viral polyprotein into
number of essential enzymes (reverse transcription,
polymerase).
•
Examples are : Saquinavir, and Ritonavir.
•
They are orally active, side effects include GI
disturbances and hyperglycemia,
interact
with
cytochrome P450. buffalo hump
Anti-Viral Chemotherapy
GAG/POL polyprotein
GAG
Integrase
Polymerase
Protease
Retrovirus --- HIV
55
Anti-Viral Chemotherapy
GAG
Integrase
Polymerase
Protease folds and cuts itself free
56
Anti-Viral Chemotherapy
GAG
Integrase
Polymerase
Protease cuts at a site between the integrase and polymerase
57
Anti-Viral Chemotherapy
GAG
Integrase
polymerase
58
New targets
• Agents that block fusion of HIV with the host cell by
interacting with gp41
• Enfuvirtide is Peptides derived from gp41 can inhibit
infection, probably by blocking the interaction of gp41 with
cell membrane proteins during fusion.
• Raltegravir (Integrase Inhibitor) targets integrase, an HIV
enzyme that integrates the viral genetic material into human
chromosomes, a critical step in the pathogenesis of HIV.
• Maraviroc It blocks the interaction between chemokine
receptor CCR5 and HIV gp120.
(HAART)
• Highly active anti-retroviral therapies
• Combination therapies (triple drug cocktail, HAART) are very
effective and can reduce viral load in the patient below detectable
levels implying that HIV replication has ceased.
examples (1) NNRTI–Based Regimens (1-NNRTI + 2NRTIs)
(2) PI-Based Regimens (1 or 2 PIs + 2 NRTIs)
• The trouble with all of these complicated drug regimens is
compliance. The components of HAART must be taken at
different times.
• Non-compliance with protease inhibitor therapy is of serious
concern as the new virus that emerges is resistant to the inhibitor
being taken and also resistant to other protease inhibitors.
Figure 2. Timeline of evolution of HBV therapies, United States.
Anti-Hepatitis B Virus Agents
Interferons
• Interferon Alfa
• Endogenous proteins induce host cell enzymes that inhibit viral RNA
translation and cause degradation of viral mRNA and tRNA .
• Bind to membrane receptors on cell surface , May also inhibit viral
penetration, uncoating, mRNA synthesis, and translation, and virion
assembly and release.
• Pegylated interferon Alfa
• A linear or branced polyethylene gylcol (PEG) moiety is attached
covalently to interferon
• Increased
half-life
and
steady
drug
concentrations
Interferon, mechanism of action:
• 1) binds to cell surface receptors
• 2) induces expression of translation inhibitory
protein (TIP)
• 3) TIP binds to ribosome, inhibits host
expression of viral proteins
Interferons
•
a limited treatment course (ie, only 1 year of therapy),
•
lack of resistance development.
• Disadvantages include a high rate of treatment-related
adverse events. flu-like symptoms: increased body
temperature, feeling ill, fatigue, headache, muscle pain.
Anti-Hepatitis B Virus Agents
• …….
• Entecavir and tenofovir have very strong resistance profiles
in treatment-naive patients.
• Disadvantages include the need to continue therapy
indefinitely and the potential for resistance development.
Anti-Hepatitis C Virus Agents
• Approved
– Interferon-alpha (pegylated)
– Ribavirin
• In development
– Protease inhibitors
– Polymerase inhibitors
• In pregnancy , a regimen of oral zidovudine
beginning between 14 and 34 weeks of
gestation, intravenous zidovudine during
labor, and zidovudine syrup to the neonate
from birth through 6 weeks of age has been
shown to reduce the rate of vertical (motherto-newborn) transmission of HIV by up to
23%.
Kirby-Bauer Method for Determining Drug
Susceptibility
1. Bacteria spread on surface of agar plate
2. 12 disks, each with different antimicrobial
drug, placed on agar plate
3. Incubated- drugs diffuse outward and kill
susceptible bacteria
4. Zone of inhibition around each disk
5. Compare size of zone to chart
Figure 21.10
Resistance to Antimicrobial Drugs
• Drug resistance limits use of ALL known
antimicrobials
• Penicillin G: first introduced, only 3% of
bacteria resistant
• Now, over 90% are resistant
Mechanisms Responsible for Resistance to
Antimicrobial Drugs Include the Following:
1. Inactivating enzymes that destroy the drug
(e.g., β-lactamases).
2. Decreased drug accumulation (e.g., tet).
3. Altering the binding sites (e.g.,
aminoglycosides and erythromycin).
4. Development of alternative metabolic
pathways (sulphonamides ( dihydropteroate
synthease) and trimethoprim (dihydrofolate
reductase).
How do Bacteria Become Resistant?
1. Spontaneous Mutation: happen as cells
replicate – Within a pop, there will be some
bact with acquired resistance. The drug
then elim the sensitive organisms, while the
resistant ones proliferate.
2. Gene Transfer or Transferred resistance:
Usually spread through conjugative transfer
of R plasmid ( may be virally mediated).
Slowing the Emergence and Spread of
Antimicrobial Resistance
1. Responsibilities of Physicians: must work to
identify microbe and prescribe suitable
antimicrobials, must educate patients
2. Responsibilities of Patients: need to carefully
follow instructions
Slowing the emergence and spread of
antimicrobial resistance
3. Educate Public: must understand
appropriateness and limitations of antibiotics;
antibiotics not effective against viruses
4. Global Impacts: organism that is resistant can
quickly travel to another country
- in some countries antibiotics available on
non-prescription basis
- antibiotics fed to animals can select for drugresistant organisms
New Approaches to Antibiotic Therapy Are
Needed
• Scientists work to find new antibiotic targets
in pathogens
• Discovery of new and unique antibiotics is
necessary